This project will develop a super resolution pump-probe modulation (PPM) microscope to image non-fluorescent molecules. Imaging has become an important tool in biomedical research and clinical prognosis and treatment. Optical imaging systems, particularly fluorescence microscopy, have been extensively used in biomedical research. Recently, stimulated emission depletion (STED) microscopy has achieved nanometer resolution using point-spread function (PSF) engineering techniques to break the diffraction limit. All these super resolution techniques are based on molecular fluorescence with most applications requiring tagging exogenous fluorophores onto targeted molecules. However, the labeling process can alter the activity or localization of the molecules. Recently pump-probe microscopy demonstrated its ability to image non-fluorescent molecules by detecting the energy transitions between different molecular energy levels when two femtosecond/picosecond laser pulses are interacting with the molecules. The common feature of the pump-probe microscope and the STED microscope is that both modalities use two laser beams to excite and then to probe or deplete the molecules. The innovation of this proposal is developing a pump-probe modulation (PPM) microscope that combines the PSF engineering method used in STED microscopy and the pump-probe method to achieve the goal of imaging non-fluorescent molecules with nanometer resolution. There are two specific aims in this grant application: (1) Develop a pump-probe modulation microscope based on stimulated emission and ground state depletion that can image gold nanorods and Atto612Q molecules with super resolution; and (2) Image dopamine in live Drosophila brain tissue with two-photon excitation PPM microscopy.